Abstract：
A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.
摘要：
流感嗜血杆菌中氯霉素敏感性降低通常是由氯霉素乙酰转移酶（CAT）的活性引起的。然而,在流感嗜血杆菌的氯霉素敏感性中,膜蛋白的参与仍不清楚.在这项研究中,氯霉素药敏试验,全基因组测序，并对51株流感嗜血杆菌分离株的膜相关基因进行了分析。进行功能互补测定和基于结构的蛋白质分析以评估具有序列取代的蛋白质对CAT阴性流感嗜血杆菌分离物中氯霉素的最小抑制浓度(MIC)的影响。6个分离株对氯霉素耐药，对A-2型CAT呈阳性。在这些分离物中，相对于其他抗性分离株，A3256具有相似的CAT活性水平，但氯霉素MIC更高；它在58个膜基因中也有163个特定变异。关于CAT阴性分离株，逻辑回归和受试者算子特征曲线分析显示48T>G(Asn16Lys)，85C>T(Leu29Phe)，和88C>A(Leu30Ile)在HI_0898(emrA)中，HI_1177（artM）中86T>G（Phe29Cys）和141T>A（Ser47Arg）与氯霉素敏感性增强有关，而HI_1612（hmrM）中的997G>A（Val333Ile）与氯霉素敏感性降低有关。此外，在具有EmrA-Leu29Phe/Leu30Ile或ArtM-Ser47Arg取代的CAT阴性分离株中，氯霉素MIC较低，而在具有HmrM-Val333Ile取代的分离株中，相对于他们的同行。Val333Ile取代与CAT阴性流感嗜血杆菌分离株中HmrM蛋白稳定性和灵活性增强以及氯霉素MIC增加相关。总之,流感嗜血杆菌多药外排泵HmrM的替代与氯霉素敏感性降低相关。
